1. Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver
- Author
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Martin Bidlingmaier, Birgit Rathkolb, Martin Hrabĕ de Angelis, Arne Hinrichs, Werner F. Blum, Sebastian Bultmann, Heinrich Leonhardt, Maik Dahlhoff, Simone Renner, Andreas Blutke, Eckhard Wolf, Barbara Kessler, Andreas Hoeflich, Hiroshi Nagashima, Maren Bernau, Mayuko Kurome, Rüdiger Wanke, Elisabeth Kemter, and Armin M. Scholz
- Subjects
0301 basic medicine ,Insulin-like growth factor 1 ,Swine ,INSR, insulin receptor ,medicine.medical_treatment ,4EBP1, eukaryotic initiation factor 4E binding protein 1 ,IGFBP3 ,Dwarfism ,IGF1, insulin-like growth factor 1 ,Growth hormone receptor ,PCR, polymerase chain reaction ,LDL, low-density lipoprotein ,Laron syndrome ,STAT5 Transcription Factor ,Insulin-Like Growth Factor I ,LSM, least squares mean ,CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated ,JAK2, Janus kinase 2 ,Adiposity ,mTOR, mechanistic target of rapamycin ,HOMA, homeostatic model assessment ,DXA, dual-energy X-ray absorptiometry ,ELISA, enzyme-linked immunosorbent assay ,Pig model ,eIF4E, eukaryotic translation initiation factor 4E ,AKT, serine-threonine protein kinase ,PPARG, peroxisome proliferator-activated receptor gamma ,STAT, signal transducer and activator of transcription ,GHR, growth hormone receptor ,LPL, lipoprotein lipase ,Liver ,mTORC, mTOR complex ,S6K, protein S6 kinase 1 ,Original Article ,IRS1, insulin receptor substrate 1 ,Growth Hormone Receptor ,Laron Syndrome ,Pig Model ,Hypoglycemia ,Insulin-like Growth Factor 1 ,Signaling ,HSL, hormone-sensitive lipase ,PI3K, phosphoinositide 3 kinase ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction ,medicine.medical_specialty ,lcsh:Internal medicine ,aa, amino acid ,HDL, high-density lipoprotein ,IgG, immunoglobulin G ,Mechanistic Target of Rapamycin Complex 2 ,Biology ,03 medical and health sciences ,GSK3B, glycogen synthase 3 beta ,Internal medicine ,medicine ,Animals ,LS, Laron syndrome ,lcsh:RC31-1245 ,Molecular Biology ,Leptin receptor ,DAB, 3,3′-diaminobenzidine ,SE, standard error ,Growth factor ,Body Weight ,sgRNA, single guide RNA ,Cell Biology ,Receptors, Somatotropin ,Janus Kinase 2 ,medicine.disease ,IRS1 ,GH, growth hormone ,TBS, Tris-buffered saline ,Insulin receptor ,Insulin-Like Growth Factor Binding Protein 2 ,AMPK, AMP-activated protein kinase ,030104 developmental biology ,Endocrinology ,Insulin-Like Growth Factor Binding Protein 3 ,IGFBP, IGF-binding protein ,LEPR, leptin receptor ,Growth Hormone ,biology.protein ,RIA, radioimmunoassay ,MRI, magnetic resonance imaging ,MAPK, mitogen-activated protein kinase - Abstract
Objective Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized. Methods CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue. Results GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2. Conclusion GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials., Highlights • GHR-deficient pigs reveal postnatal growth retardation, disproportionate organ growth and an increased total body fat content. • GHR-deficient pigs show markedly reduced serum IGF1 and IGFBP3 levels, and transient juvenile hypoglycemia. • Increased expression and phosphorylation of IRS1 in liver of adult GHR-deficient pigs suggest increased insulin sensitivity. • Increased phosphorylation of JAK2 in liver of GHR-deficient pigs may be explained by higher serum leptin levels and activation of hepatic LEPR.
- Published
- 2018